European Journal of Applied Physiology

, Volume 90, Issue 5, pp 639–642

Blood coagulation and fibrinolysis after long-duration treadmill exercise controlled by individual anaerobic threshold

Authors

    • Department of Sports MedicineFriedrich-Schiller-University Jena
  • Doreen Gläser
    • Department of Sports MedicineFriedrich-Schiller-University Jena
  • Carsten Reckhart
    • Department of Sports MedicineFriedrich-Schiller-University Jena
  • Dagmar Prasa
    • Centre of Vascular Biology and Medicine, ErfurtFriedrich-Schiller-University
  • Jörg Stürzebecher
    • Centre of Vascular Biology and Medicine, ErfurtFriedrich-Schiller-University
  • Holger H. W. Gabriel
    • Department of Sports MedicineFriedrich-Schiller-University Jena
Short Communication

DOI: 10.1007/s00421-003-0907-2

Cite this article as:
Hilberg, T., Gläser, D., Reckhart, C. et al. Eur J Appl Physiol (2003) 90: 639. doi:10.1007/s00421-003-0907-2

Abstract

For rehabilitation training it is recommended that the intensity of exercise should be clearly below the individual anaerobic threshold (IAT). We investigated blood coagulation, particularly endogenous thrombin potential (ETP) and fibrinolysis following a standardized treadmill (TR) ergometer test at 90% IAT for 60–120 min. Sixteen healthy male non-smokers underwent the TR test. Blood samples were taken after a 30-min rest, immediately after exercise, and 2 h after exercise completion. Extrinsic and intrinsic total (TTPex+in) and endogenous (ETPex+in) thrombin potential, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), plasmin-α2-antiplasmin complex (PAP), D-dimer, tissue plasminogen activator antigen and activity (tPA-AG and tPA-ACT) and plasminogen activator inhibitor type 1 antigen and activity (PAI-1-AG and PAI-1-ACT) were measured. Immediately after TR, F1+2, TAT and TTPex+in were increased (P<0.05) while ETPex+in remained unchanged. In contrast, PAP, D-dimer, tPA-AG, tPA-ACT (P<0.05) were distinctly enhanced while PAI-1-ACT was decreased (P<0.05) immediately after exercise. The changes in tPA-AG, tPA-ACT, and PAI-1-ACT were reversed to nearly baseline while the enhancement in PAP and D-dimer was prolonged by more than 2 h after exercise. Long-duration exercise between 60 and 120 min controlled by IAT (90%) on a TR ergometer only implicates a small increase in thrombin generation markers and total (free and α2-macroglubulin-bound thrombin), but not in endogenous (free) thrombin potential alone. In contrast, fibrinolysis is distinctly increased after this type of exercise. Endurance exercise with an intensity below 90% IAT and a duration below 2 h generates a more favourable condition for fibrinolysis than for blood coagulation in healthy young subjects. Data are given as mean (SD).

Keywords

Endogenous thrombin potentialPhysical activityThrombin potentialTissue plasminogen activatorTreadmill

Introduction

Exhaustive exercise may increase the risk of cardiac syndromes or cardiovascular events by activation of blood coagulation or platelet activity, but endurance exercises, e.g. with intensities distinctly below the individual anaerobic threshold (IAT) are recommended for rehabilitation training. Weiss et al. (1998) showed that thrombin generation markers are enhanced after heavy (83% V̇O2max) but not after moderate (68% V̇O2max) treadmill exercise for 1 h. Hegde et al. (2001) have suggested that submaximal running for 1 h (70–75% V̇O2max) implicates a "more favourable situation" for clot formation after exercise.

Too little is known about changes in haemostasis after exercises, controlled by the IAT. The IAT is a commonly used method to control exercise intensity; at IAT lactate production and elimination are in a steady state but above this point lactate concentration increases in whole blood leading to exhaustion of the subject (Urhausen et al. 1994). By determining the IAT, the hypothesis that a moderate but long-duration standardized treadmill exercise with a duration of 60–120 min and 90% IAT does not enhance blood coagulation, but triggers fibrinolysis, can be investigated.

For the determination of blood coagulation an additional method was used in this study. Hemker et al. (1993) described a method for determination of endogenous thrombin potential (ETP). This parameter gives information about how much thrombin can be generated in plasma after activation of the extrinsic and intrinsic coagulation pathway. Thus, the ETP is a measure for the functionality of the coagulation system indicating hyper- or hypocoagulant states.

The aim of the study was to investigate the changes in blood coagulation measured by thrombin generation markers and thrombin potential, and fibrinolysis after a long-duration standardized treadmill ergometer test with an intensity of 90% IAT, lasting 60–120 min in healthy men.

Methods

Subjects

The subjects (16 healthy male non-smokers) had an average age of 25 (5) [mean (SD)] years. Mean weight was 77 (8) kg, mean height 183 (6) cm, and mean body fat 11.0 (2.5)%). The mean V̇O2 peak (step test; treadmill ergometer) was 57.3 (4.5) ml kg−1 min−1 determined by the Oxycon beta (Jaeger, Hoechberg, Germany), mean maximal power was 4.6 (0.3) m s−1, mean IAT 3.4 (0.3) m s−1, and mean heart volume 10.8 (0.8) ml kg−1. Procedures used in this study were approved by the Ethics Committee of the Faculty of Medicine of the Friedrich-Schiller-University Jena. Written informed consent was obtained from each subject, prior to the start of the study.

Maximal exercise test

One to 2 weeks before the test program all subjects performed an incremental graded exercise on a treadmill ergometer (step test; start 2 m s−1, every 3 min an increase of 0.5 m s−1 until volitional exhaustion) to measure V̇O2 peak and the IAT, determined according to the method of Stegmann et al. (1981). Capillary blood samples were obtained from the ear lobe at rest, at the end of each level of exercise, and at the end of the 1st, 3rd, 5th, and 10th min of the recovery period. Maximal lactate concentration measured by the EBIO plus (Eppendorf, Hamburg, Germany) was 9.0 (1.8) mmol l−1.

Exercise program

The standardized treadmill exercise test was initiated after at least a 10-h overnight fast with the exception of a standardized low-fat breakfast in the morning. The test included an intensity of 90% IAT and the subjects were asked to realize an exercise duration of a minimum of 60 min until individual exhaustion. After 120 min exercise was stopped by the investigator. The mean duration of the exercise was 96 (22) min, with a mean speed of 3.1 (0.3) m s−1 and a completed distance of 17.9 (4.7) km. Mean lactate before exercise was 1.68 (0.48) and after exercise 3.29 (1.01) mmol l−1. All the exercise tests were done between 8.00 a.m. and 1.00 p.m. in the laboratory.

Analytical methods

Blood sampling and laboratory methods

Blood samples were taken by a clean venipuncture (20-gauge needle) from an antecubital vein under controlled venous stasis (<30 s) of 40 torr after 30 min rest, immediately after, and 2 h after exercise. Samples were collected in the following sequence, after discarding the first 3 ml blood: (1) 2.61 ml of blood was added to 0.29 ml of CTAD solution (Sarstedt, Nümbrecht, Germany) for the assessment of plasminogen activator inhibitor type 1 antigen (PAI-1-AG; Coaliza PAI, Chromogenix Instrumentation Laboratory; Milano, Italy); (2) 4.5 ml of blood was added to 0.5 ml of stabilyte solution (citric acid and trisodium citrate, pH 4.3; Sarstedt) for the assessment of tissue plasminogen activator antigen and activity (tPA-AG, tPA-ACT), and PAI-1-ACT (Coaliza t-PA, Coaset t-PA, Coatest PAI; Chromogenix Instrumentation Laboratory); (3) 9 ml of blood was added to 1 ml of 0.106 M trisodium citrate for the assessment of prothrombin time (PT), activated partial thromboplastin time (aPTT), which were done on ACL 7000 (IL), and prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin III complex (TAT) via ELISA (Enzygnost F1+2micro and Enzygnost TATmicro; Dade Behring, Marburg, Germany) and plasmin-α2antiplasmin complex (PAP) and D-dimer via Enzygnost PAP micro and Enzygnost D-dimer (Dade Behring), and haematocrit (Act-Diff; Coulter Electronics, Krefeld, Germany). The ELISAs were measured on a microplate reader Dynatech MR 4000 (Dynex, Denkendorf, Germany); (4) 9 ml of blood was added to 1 ml of 0.106 M trisodium citrate for assessment of thrombin generation as explained in the following section.

Multiple aliquots of plasma were snap-frozen and stored at –80°C until analysis. All samples from the same athlete were measured at the same assay and as duplicates. Intra-assay coefficients of variation (CV) were below 8% and inter-assay coefficients below 15% for the ELISA tests.

Changes in plasma volume were calculated for F1+2, TAT, PAP, D-dimer, tPA-AG and PAI-1-AG as described in the literature (Dill and Costill 1974).

Measurement of thrombin generation

On a microtitre plate, 200 μl plasma was mixed with 25 μl TRIS buffer (0.05 M, NaCl 0.1 M, HSA 0.5%; pH 7.4) and 30 μl of the chromogenic substrate H-βAla-Gly-Arg-pNA (5 mM; Pefachrome TG; Pentapharm, Basel, Switzerland). To block fibrin polymerization during measurement, 25 μl Pefabloc FG (36 mg ml−1; Pentapharm) was added. After prewarming to 37°C the plasma was activated either with 20 μl Innovin (in 0.25 M CaCl2; Dade Behring) or with 20 μl DAPTTIN (in 0.25 M CaCl2; Baxter, Vienna, Austria). The release of pNA was measured at 405 nm during a period of about 16 min at intervals of 10 s with the microplate reader iEMS-MF (Labsystems, Finland). The first derivation of the curve of OD traces and with it the thrombin activity were calculated by a computer program according to Hemker et al. (1993) considering the concentrations and the kinetic constants of the substrate. The area under the curve (total thrombin potential, TTP) represents the total amount of thrombin generated in plasma including both free and α2-macroglobulin-bound thrombin (α2M-IIa). After mathematical subtraction of α2M-IIa, the area under this curve represents the endogenous (free) thrombin potential (ETP). All samples of the same athlete were measured at the same time and as duplicates. Intra-assay coefficients of variation (CV) were below 3% and inter-assay coefficients below 4% for these tests.

Statistics

Results are reported as mean (SD). The data did not show normal distribution, which was tested by the Kolmogorov-Smirnov-test; therefore, the Wilcoxon-rank-test was used for testing the changes before and after exercise. The level of significance was set at P<0.05. Statistical analysis was done with SPSS-10.0 software.

Results

Blood coagulation

After the exercise a significant (P<0.05) shortening of aPTT by 14% was seen. This was not totally reversed 2 h after exercise. In addition TAT and F1+2 showed a higher (P<0.05) concentration by 53% and 34%, respectively, after exercise and TAT but not F1+2 was reversed to near baseline after 2 h. Intrinsic and extrinsic total thrombin potential was also higher (P<0.05) immediately after exercise but the intrinsic and extrinsic ETP remained unchanged (Table 1).
Table 1.

Blood coagulation before (pre-), immediately after (post-) and 2 h post-exercise. aPTT activated partial thromboplastin time, ETPin/ex intrinsic/extrinsic endogenous thrombin potential, F1+2 prothrombin fragment 1+2, PT prothrombin time, TAT thrombin-antithrombin complex, TTPin/ex intrinsic/extrinsic total thrombin potential

Pre-exercise

Post-exercise

2 h post-exercise

PT (%)

Mean (SD)

96 (13)

96 (13)

88 (14)* **

Range

71–116

69–113

65–110

aPTT (s)

Mean (SD)

32.9 (3.4)

28.2 (3.1)*

29.7 (3.4)*

Range

27.5–39.7

21.6–35.2

23.0–37.4

TAT (μg l−1)

Mean (SD)

2.25 (1.16)

3.45 (1.03)*

2.32 (0.43)**

Range

1.40–6.47

2.00–6.36

1.69–3.18

F1+2 (nmol l−1)

Mean (SD)

0.64 (0.17)

0.86 (0.23)*

0.74 (0.20)* **

Range

0.35–1.06

0.53–1.42

0.42–1.18

TTPin (nmol min−1 l−1)

Mean (SD)

1668 (224)

1786 (272)*

1576 (286)* **

Range

1184–2117

1354–2257

1041–1985

TTPex (nmol min−1 l−1)

Mean (SD)

2159 (340)

2304 (441)*

2120 (359)**

Range

1587–2838

1538–2951

1480–2699

ETPin (nmol min−1 l−1)

Mean (SD)

651 (145)

630 (106)

617 (127)

Range

379–898

500–831

381–811

ETPex (nmol min−1 l−1)

Mean (SD)

652 (138)

674 (137)

646 (137)

Range

365–940

352–905

313–937

*P<0.05, significant to pre-exercise; **P<0.05, significant to post-exercise

Fibrinolysis

PAP was distinctly increased (P<0.05) by 582% after exercise and failed to return to baseline after 2 h. A similar statistically relevant change was observed in D-dimer which was enhanced (P<0.05) by 76% immediately afterwards and persisted for 2 h after exercise. TPA-AG as well as TPA-ACT were both increased (P<0.05) after exercise and returned to near baseline after 2 h. In contrast PAI-1-AG remained unchanged and PAI-1-ACT distinctly decreased (P<0.05) immediately after exercise (Table 2).
Table 2.

Fibrinolysis before (pre-), immediately after (post-) and 2 h post-exercise. ACT activity, AG antigen, AU arbitrary unit, PAI–1 plasminogen activator inhibitor type 1, PAP plasmin-α2-antiplasmin complex, tPA tissue plasminogen activator

Pre-exercise

Post-exercise

2 h post-exercise

PAP (μg l−1)

Mean (SD)

321 (120)

2190 (976)*

853 (353)* **

Range

109–511

923–4372

374–1639

D–dimer (μg l−1)

Mean (SD)

10.4 (7.1)

18.3 (12.7)*

16.4 (12.9)* **

Range

3.7–32.5

7.3–53.1

4.9–54.6

tPAAG (ng ml−1)

Mean (SD)

2.7 (1.2)

22.6 (6.0)*

4.0 (2.1)**

Range

1.3–5.9

12.3–32.1

1.6–9.1

tPAACT (IU ml−1)

Mean (SD)

6.9 (1.8)

9.1 (5.3)*

6.3 (1.4)**

Range

4.3–9.7

5.6–27.2

3.6–8.1

PAI–1AG (ng ml−1)

Mean (SD)

37.7 (15.1)

34.6 (11.0)

43.6 (16.2)**

Range

22.0–74.4

18.6–55.3

22.0–85.1

PAI–1ACT (AU ml−1)

Mean (SD)

11.2 (4.1)

3.5 (2.9)*

12.9 (2.3)**

Range

2.2–16.3

0.0–7.6

9.2–17.4

*P<0.05, significant to pre-exercise; **P<0.05, significant to post-exercise

Discussion

Exercises of moderate intensity are recommended in rehabilitation training. Endurance training can be controlled by different methods such as oxygen consumption or IAT. Meyer et al. (1999) have shown that levels of oxygen consumption and heart rate vary considerably in relation to the IAT, and suggest that reliance on these parameters without determination of the IAT is not sufficient. Therefore, in the present study an intensity of 90% IAT for 60–120 min was used to investigate the effects on blood coagulation and fibrinolysis in healthy young non-smokers. To investigate if a hypercoagulative potential exists, a method described by Hemker et al. (1993) for the determination of an increase in ETP after exercise was also used.

In the present study an enhancement of the thrombin generation markers (F1+2 and TAT) after exercise was observed, similar to the increase described by Weiss et al. (1998) after heavy exercise but lower than described by Prisco et al. (1998) in subjects after a marathon race. In addition, the extrinsic and intrinsic TTP were increased which represents an increase of free- and α2-macroglubulin-bound thrombin potential but the ETP alone remained unchanged. This confirmed that no hypercoagulative stage existed after this type of exercise. In addition fibrinolysis was distinctly activated after this type of exercise. PAP showed a marked increase immediately afterwards and persisted for over 2 h after exercise. TPA-AG offered a higher change but tPA-ACT was also enhanced while the PAI-1-ACT was decreased immediately after exercise. In addition, the change in D-dimer confirmed a fibrin degradation in vivo after exercise. Together, these effects indicate that the balance of blood coagulation and fibrinolysis is distinctly shifted in favour of fibrinolysis after this type of exercise in healthy subjects. It would be helpful in future if patients, e.g. with cardiovascular diseases, were also investigated.

Conclusion

A long-duration standardized treadmill exercise (60–120 min) with an intensity of 90% IAT in healthy non-smokers does not lead to a higher potential for blood coagulation but clearly activates fibrinolysis. Therefore, exercising clearly below the IAT and up to 120 min generates a more favourable situation for fibrinolysis compared to blood coagulation in healthy young subjects.

Acknowledgements

The authors thank Mrs. B. Tauch and Ms. Kley for their excellent technical assistance, Mrs. I. Schellenberg for her excellent measurements of TTP and ETP, and Mr. T. Noll, Dade Behring, Marburg, Germany for his support with ELISA kits. The authors declare that the experiments comply with the current laws of Germany.

Copyright information

© Springer-Verlag 2003